Objective. To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1β (IL-1β) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. Methods. IL-1β, tumor necrosis factor α (TNFα) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4-5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green-based quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B-expressing and IL1B-nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (-299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1β into the culture media was assessed by enzyme-linked immunosorbent assay. Results. Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100-1,000-fold by treatment with TNFα/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1β and was almost abolished by TNFα/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. Conclusion. These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes.